CCL2, also known as Monocyte Chemoattractant Protein 1 (MCP-1), belongs to the CC chemokine family, which is characterized by the ability to attract mononuclear cells to sites of chronic inflammation. CCL2 is a potent chemoattractant of monocytes, and this seems to be its predominant physiological role. CCL2 has also been shown to attract dendritic and memory T cells, as well as basophils, and stimulate histamine release from the latter (Rollins, 1996, Mol Med Today. 2:198).
CCL2 is thought to play a critical role in inflammatory diseases that involve mononuclear cell infiltration, such as atherosclerosis, rheumatoid arthritis and multiple sclerosis. Furthermore, this chemokine has also been implicated in inflammation of the nervous system and other diseases and conditions, both with and without an obvious inflammatory component including atopic dermatitis, renal disease, pleurisy, allergic inflammation and asthma, colitis, endometriosis, polymyositis and dermatomyositis, uveitis, and various vascular disorders (such as atherosclerosis, restenosis after coronary intervention, intimal hyperplasia, arteriogenesis, ischemia and stroke) and even in the control of leptin secretion and hence, obesity and cachexia (Rollins, 1996, Mol Med Today. 2:198; Dawson et al., 2003, Expert Opin Ther Targets. 7:35; Daly and Rollins, 2003, Microcir. 10: 247; Charo and Taubman, 2004, Circ Res. 95: 858)
The cognate receptor for CCL2 is the seven-transmembrane-spanning G-protein-coupled receptor CCR2. The two isoforms of this receptor are CCR2A and CCR2B, which differ in their signaling C-terminal regions. The CCR2B isoform is more abundantly expressed on monocytes, and is probably the primary mediator of monocyte responses to CCL2 during inflammation (Tanaka et al, 2002, Biochem Biophys Res Commun. 290: 73). In addition to its interaction with CCR2, CCL2 binds to glycosaminoglycan (GAG) and form oligomers which are essential for its in vivo function. Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high-localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration (Proudfoot, A. E., et al., 2003, Proc Natl Acad Sci USA. 100:1885; Lau, E. K., et al., 2004, J Biol Chem. 279:22294).
The expression of CCL2 can be induced in various tissues in response to different types of inflammatory lesions. CCL2 mediates the influx of inflammatory macrophages, derived from peripheral blood monocytes, into the sites of injury. These macrophages secrete proinflammatory cytokines, tissue degrading enzymes and chemokines that mediate the influx of other inflammatory cells, leading to tissue destruction in chronic inflammatory diseases. This suggests that inhibition of monocyte migration into inflammatory lesions might be an effective mechanism to modulate disease progression in chronic inflammation (Dawson et al., 2003, Expert Opin Ther Targets. 7:35; Daly and Rollins, 2003, Microcir. 10: 247)
The critical role of the CCL2-CCR2 interaction, in modulating the tissue influx of monocytes, has been demonstrated in mice engineered to be deficient in either CCR2 or CCL2. These mice show a selective defect in the migration of macrophages to sites of inflammation (Boring, L., J. et al., 1997, J Clin Invest. 100:2552; Kurihara, T., G. et al., 1997, J Exp Med. 186:1757; Kuziel, W. A., et al 1997, Proc Natl Acad Sci USA. 94:12053; Lu, B., et al., 1998, J Exp Med. 187:601) Maus et al 2005 Am. J. Physiol. Lung Cell. Mol. Physiol. 288: L350-L358; Maus et al 2002; Am. J. Respir. Crit. Care Med. 16: 268). Furthermore, when subjected to disease induction, both CCR2 and CCL2 knockout mice are protected from inflammatory diseases, such as experimental autoimmune encephalomyelitis (EAE; a mouse model for multiple sclerosis) (Huang, D. R., et al., 2001, J Exp Med. 193:713; Izikson, L. R. et al 2000, J Exp Med. 192:1075), neuropathic pain (Abbadie, C., et al., 2003, Proc Natl Acad Sci USA. 100:7947.), and atherosclerosis (Boring, L., et al., 1998, Nature 394:894).
Chemokines other than CCL2 are agonists for CCR2, and CCL2 may act through receptors other than CCR2. However, in contrast to the redundancy suggested by in vitro studies, in vivo studies using transgenic and knockout models suggest non-redundant roles for CCL2 and CCR2 and a predominantly monocytic chemoattractant role.
CCL2 has been implicated in atherogenesis and in the formation of intimal hyperplasia after arterial injury. It is secreted by endothelial and arterial smooth muscle cells in response to vascular insults, such as hyperlipidemia. This chemokine attracts circulating blood monocytes, which accumulate in early atherosclerotic lesions in the subendothelium, differentiate into macrophages, continue to take up lipids and become foam cells of the fatty streak. CCL2 or CCR2 deficiency, or treatment with antagonists such as 7ND, provided substantial protection against plaque formation (Daly and Rollins, 2003, Microcir. 10: 247; Charo and Taubman, 2004, Circ Res. 95: 858).
CCL2 appears to play a major role in the pathogenesis of Multiple Sclerosis (MS), which involves the infiltration of effector mononuclear cells into the central nervous system. Furthermore, there is a correlation between CCL2 expression and disease activity in MS patients. CCL2 and CCR2 are not necessary for mounting an immune response to myelin antigens, but they are rather required for attracting effector cells into the CNS where they can initiate the process of demyelination and axonal severing that are characteristic of EAE and MS (Daly and Rollins, 2003, Microcir. 10: 247; Mahad and Ransohoff, 2003, Semin. Immunol. 15: 23).
CCR2 and CCL2 contribute to trafficking of macrophages and dendritic cells, an indispensable component of the host response to infectious diseases. Recruitment of T cells to sites of infection is dependent upon a functional CCR2 receptor of the antigen presenting cells (APCs). Several studies with CCR2-deficient mice indicate that the decreased recruitment of such cells leads to a functional Th1-type defect, and to increased susceptibility to various pathogens. CCL2-deficient mice, however, do not have a Th1-type defect, but rather exhibit a Th2-type impairment. These differences can be explained by the redundancy of activation of CCR2 by other MCP cytokines. CCL2 seems to have a direct effect on activated and memory T cells, suggesting its involvement in promoting Th2-type responses. Taken together, these studies support important roles for CCR2 and CCL2 in both the innate and the adaptive immune responses (Charo and Peters, 2003, Microcirc. 10: 259).
Several studies have been published using antagonistic agents to test the potential therapeutic benefits of CCR2 inhibition. The mature human MCP-1, as secreted from cells, has 76 amino acids (Rollins, 1989, Mol. Cell. Biol. 9:4687). Protein antagonists of CCR2, generated by N-terminal truncation of CCL2, namely 7ND (lacking amino acids 2-8 of the mature human MCP-1) and MCP-1(9-76), have been tested in several animal models, and were shown to inhibit experimental restenosis, intimal hyperplasia, pulmonary hypertension, atherosclerosis, focal brain ischemia and ischemia-reperfusion injury, renal fibrosis and injury, nephritis of lupus model mice, renal fibrosis, ischemia-induced neovascularization, hepatic fibrosis, experimental autoimmune myocarditis, and arthritis (Kitamoto and Egashira, 2002, J. Atheroscler. Thromb. 9: 261; Kitamoto and Egashira, 2003, Expert Rev. Cardiovas. Ther. 1: 393).
An antibody generated against murine CCR2 (MC-21) reduces the in vitro activity of MCP-1 by 95%, and almost completely prevented the influx of monocytes in a murine model of acute peritonitis (Mack et al 2001 J. Immunol. 166: 4697). Systemic administration of this antibody was effective also in blocking alveolar monocyte recruitment in response to lung deposition of CCL2 and/or LPS (Maus et al 2005 Am. J. Physiol. Lung Cell. Mol. Physiol. 288: L350-L358; Maus et al 2002, Am. J. Respir. Crit. Care Med. 166: 268). An anti-human CCR2 was effective in a primate model of experimental in-stent restenosis (Horvath et al, 2002, Circ. Res. 90: 488).
There is a growing interest in CCR2 antagonists and hence there are several inhibitors that target the CCR2/CCL2 system which are currently in clinical development including small organic molecules as well as antibodies. For example, INCB3284, Incyte's most advanced CCR2 antagonist, which is currently in Phase IIa clinical trials in rheumatoid arthritis and obese insulin-resistant patients. An additional CCR2 antagonists in Phase II clinical trials are Merck's MK0812, for treatment of relapsing-remitting multiple sclerosis, and Millennium's MLN-1202, a humanized monoclonal antibody specific for CCR2, for the treatment multiple sclerosis, atherosclerosis and scleroderma. Several other agents that target the CCR2/MCP-1 system are in earlier stages of development. For example, CCX915, an orally available CCR2 antagonist is in Phase I development by ChemoCentryx, for treatment of multiple sclerosis. In addition, Telik has an MCP-1 antagonist in preclinical development for treatment of rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, atherosclerosis, asthma, and cancer.
WO 05/072049 assigned to the applicant of the present invention discloses polynucleotides and their respective encoded polypeptides and assays and methods of use thereof in the diagnosis of endometriosis. One among the hundreds of polynucleotide transcripts disclosed therein is S71513_T2 (denoted herein SEQ ID NO:1) which encodes an amino acid sequence denoted S71513_P2 (denoted herein SEQ ID NO:9), which is disclosed herein to correspond to a splice variant of MCP-1. There is no teaching or suggestion of any utility other than for endometriosis.
Various point mutations of MCP-1 have been studied. Notably, Beall et al. (J. Biol. Chem. 267, 3455-3459, 1992) reported that a double mutation of tyrosine 28 and arginine 30 to leucine and valine, denoted Y28L and R30V respectively, causes a dramatic decrease in chemoattractant activity toward monocytes with the appearance of a novel (IL-8 like) neutrophil chemotactic activity.
U.S. Pat. No. 5,459,128 to Rollins et al. discloses additional point mutations and other variants of MCP-1, including N terminal and C terminal deletions. Mutated MCP-1 derivatives were tested for their ability to inhibit monocyte chemotaxis in vitro in response to non-mutated MCP-1. Only certain point mutations and the N terminal deletion variant denoted 7ND were able to inhibit monocyte chemotaxis. Inhibitory activity was specific for MCP-1 since the mutations did not inhibit monocyte chemotaxis in response to other chemoattractants. Manipulations of the C-terminal α-helix produced proteins that were still able to signal, but had reduced potency compared to wild-type. Deletions of half (D2) or all (D1) of the α-helix yielded proteins with 17% and 11.3% of wild-type activity, respectively. Thus, the findings of this disclosure are teaching away from C terminal deletions having MCP-1 inhibitory effects. U.S. Pat. No. 5,705,360, U.S. Pat. No. 5,739,103 and U.S. Pat. No. 5,854,412 all to Rollins et al. disclose and claim N-terminal deletion mutations of MCP-1 and other chemokines.
Thus, the background art does not teach or suggest inhibitory variants of MCP-1 protein with relatively low homology to human MCP-1 at the C-terminal portion of the molecule. The background art also does not teach or suggest therapeutic utility of MCP-1 variants with deletions or truncations of the predicted C-terminal alpha helix. The background art also does not teach or suggest that such splice variants of MCP-1 protein are useful as therapeutic proteins or peptides for a range of clinical conditions and diseases. The background art also does not teach or suggest that such splice variants of MCP-1 protein are useful as diagnostic markers and/or in diagnostic assays or methods for diagnosis of diseases.
The present invention overcomes deficiencies of the background art by providing MCP-1 therapeutic protein variants and derived peptides, which may be used as active, specific and stable therapeutic proteins or diagnostic markers.